Project 1: Genetic method for selective elimination of damaged mitochondria Mitochondrial turnover has been postulated as a mechanism for mitochondrial quality control. However, it remains a question whether cells are indeed able to eliminate defective mitochondria selectively. Quantitative and live imaging assays are required to measure selective mitochondrial degradation and visualize this process in real time, while a genetic approach is essential to probe mitochondrial turnover in a physiological context. We expressed a toxic bacterial protein, PorB, to damage a subpopulation of total cellular mitochondria in cultured Drosophila cells and tissues. Damaged mitochondria concentrated with PorB were segregated from the mitochondrial network through a fission/fusion process and selectively removed by lysosomes through the autophagy pathway in otherwise healthy cells. We demonstrated for the first time the Parkin-dependent degradation of damaged mitochondria in an animal tissue, the Drosophila flight muscle. Our work proves in principle that defective mitochondria are selectively removed in healthy cells, and also provides a novel genetic approach to monitor mitochondrial turnover and dissect the underlying mechanisms. Project 2: Selective transmission of healthy mtDNA during Drosophila oogenesis One of the most prominent questions regarding mtDNA inheritance is how mothers ensure the transmission of healthy mitochondria to their progeny. Recent studies of mtDNA mutator mice show that mtDNA mutations are purged from germline cells, even though their levels in the cells are too low to impair overall cellular fitness. The most plausible explanation for this phenomenon is that mtDNA mutations can be selected against on an organelles level based on the functionality of an individual mitochondrion. To examine this question, we turned to Drosophila oogenesis to explore the mechanisms of mitochondrial DNA transmission. We generated heteroplasmic fly lines that contain both wild type and mt:Co1T300I mtDNAs. mt:Co1T300I is a temperature sensitive mutation in the mtDNA gene cytochrome c oxidase 1 (mt:Co1). Homoplasmic mt:Co1T300I flies cannot survive at high temperature, while is mostly healthy at low temperature. We found that the frequency of mutant mtDNA in the heteroplasmic flies increased in the progeny of female flies shifted from 18C to 29C, indicating a direct selection against defective mitochondria. Cell biological analysis revealed that mtDNA replication occurs at a specialized structure in the germarium, known as the fusome, and disruption of this structure leads to a decrease in mtDNA replication early in oogenesis. Homoplasmic mt:Co1T300I flies contain an intact fusome, but display disruption of mtDNA replication in the germarium. Further, we expressed a bacterial porin protein to damage a subset of mitochondria in ovary. We observed segregation of damaged mitochondria away from the future oocyte during early and mid-stage oogenesis. Our results demonstrate that healthy mitochondria are selectively recruited to the fusome, where mtDNAs are preferentially replicated. The selective amplification of healthy mitochondria that containing wild type mtDNA will reciprocally reduce the frequency of mtDNA in ovary and their opportunity of transmission. Future studies will seek to determine the machinery involved in recognition and selection of healthy mitochondria during oogenesis. Project 3: A Drosophila model reveals novel pathogenic mechanism of mtDNA mutation. Applying a selection scheme based on mitochondrially targeted restriction enzymes, we isolated a homoplasmic mitochondria DNA mutant, mt:CoIT300I that carrying a single amino acid substitution on cytochrome C oxidase (COX) subunit I (CoI) locus. The mt:CoIT300I flies had reduced COX activity and decreased ATP levels. The mt:CoIT300I flies are temperature sensitivities. Very few survived through the pupa stage at 29 C. In addition, mt:CoIT300I flies displayed greatly reduced life span as well as impaired mobility and neural activities at permissive temperature. The defects are exacerbated in old flies, which indicates an age-dependent neurological and muscular dysfunction. Most of the phenotypes resembled typical features of human mtDNA diseases, validating mt:CoIT300I as a Drosophila model to understand the conserved features of mtDNA mutations. Expression analyses revealed handful genes that are involved in maintaining cellular redox potential and protecting against stress induced protein denaturation, are up regulated in the mutant background. Over expression of glutathione peroxidase in mt:CoIT300I flies can partially suppress the phenotype, further confirming the idea that deregulation of cellular redox potential is one of the mitochondria dysfunctions and contribute to cellular deficiencies downstream. In contrast to the common hypothesis that reactive oxygen species are one of the major players in pathogenesis induced by mitochondrial deficiencies, we found not evidence of involvement of ROS in the process in mt:CoIT300I flies. We results suggest a novel pathway of mitochondrial etiology, and provide a genetic handle to further delineate the whole process. Since most mtDNA diseases shows tissue-to-tissue variation in extent and phenotypes, we established a genetic scheme to make homoplasmic mtDNA mutation in tissue specific manner in Drosophila for a better modeling of human mtDNA diseases in future. Project 4: Targeted mutagenesis of mammalian mtDNA through direct transformation of engineered mtDNA The ability to induce specific mutations into the mammalian mitochondrial genome would facilitate studies into human mitochondrial genetics and genetic disorders. Until now, the delivery to and expression of exogenous nucleic acids in mitochondria has been limited to yeast, plants, and algal species owing to the highly active recombination of native mitochondrial genomes. In certain mammalian tissues, however, recombination of the mitochondrial DNA (mtDNA) is markedly minimal to absent. Full-length mtDNA was cloned into a plasmid and stably amplified and mutagenized in bacteria. This Engineered mtDNA plasmid was delivered to mouse fibroblast mitochondria via biolistic bombardment. The constructs were selected for using an inducible mitochondrial-targeted restriction endonuclease to which our constructs are resistant, and the 2433 T-to-C chloramphenicol resistance polymorphism. Galactose treatment assured that rho-zero cells were eliminated. Simple cell survival, along with restriction digest analysis and mtDNA fluorescent staining, indicated that our constructs had been stably integrated into mitochondria. Our preliminary evidences showed the promise of direct transformation of mammalian cell with in-vitro modified mtDNA, which could enable the study of human mtDNA disorders in animal models as well as accelerate the understanding of mtDNA genetics in mammals.